CN109750051B - Preparation of Dehydroepiandrosterone (DHEA) from 3 beta-hydroxysteroid dehydrogenase - Google Patents

Abstract

The invention relates to a novel preparation method of dehydroepiandrosterone, belonging to a preparation method of steroid compounds, which is characterized in that 5-AD is taken as a substrate, and 3-position carbonyl is reduced by 3 beta-hydroxy steroid dehydrogenase to obtain Dehydroepiandrosterone (DHEA). The method utilizes carbonyl reductase, namely 3 beta-hydroxysteroid dehydrogenase, as a catalyst, and can realize the high regioselective and stereoselective reduction of the 3-carbonyl.

Description

Preparation of Dehydroepiandrosterone (DHEA) from 3 beta-hydroxysteroid dehydrogenase

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method of Dehydroepiandrosterone (DHEA), which is realized by utilizing high regioselectivity and stereoselectivity of 3 beta-hydroxysteroid dehydrogenase.

Background

DHEA is often used as one of the steroidal drugs as an anti-aging drug because it is believed that a reduction in the level of DHEA in the body is closely related to the onset of a range of aging-related conditions. Some articles show that humans at age 80 have a DHEA content of 1/10 in their body at age 25. Dehydroepiandrosterone sulfate (DHEAS) is generally considered to be a more stable form of DHEA. DHEA and DHEAS are considered to be the most important neurosteroids in the brain (Baulieu E E, Schumacher M. procesterone as a neuroactive neurosteroid, with specific reference to the effect of the pathosterone on myelation [ J ]. Steroids,2000,65(10-11): 605-. DHEA is also a key intermediate in the metabolism of cholesterol to sex hormones (e.g., testosterone, etc.). Recent studies have furthermore shown that neurosteroids are induced in memory-enhancing (Shi J, Schulze S, Lardy H A. the effect of 7-oxo-DHEA acetate on memory in healthy and old C57BL/6 microorganism [ J ]. Steroids,2000,65(3):124-129), anxiolytic, antispasmodic (Kokate T G, Svensson B E, Rogawski M. the anticlonon activity of neurosteroid-on-stressed chloride [ J ]. J Pharmacol Exp. thermal, 1994,270(3):1223-1229), antidepressant and protective against nervous systems, in particular against oxidative damage (basal S, vaccine C, cell C11. D. 23) induced by DNA in vitro mutation of protein S, DNA in culture J. 11, DNA in culture J. 3- & S, molecular strain 18, DNA in vitro mutation of growth [ 12, D. 5, D. 35, DNA in vitro strain S, cooley D M, Glickman L T, et al.reduction in DNA damage in blue and perpheral blodelyticcells of aldehyde big after procedure with Dehydroepisteroid (DHEA) [ J ] Mutat Res-Fundam Mol Mech Mutagen,2001,480 (1): 53-62). The DHEA is an important precursor compound for synthesizing other steroid medicines in the steroid industry as an important molecule in the steroid industry after the medicinal function of the DHEA is removed. Compared with DHEA, 7 alpha-OH-DHEA has higher activity in preventing in-vitro neuronal cell from hypoxia death and greatly enhances the oxidation resistance. In addition, the hydroxylated 7 alpha-OH-DHEA has strong inhibition effect on tumor proliferation (Liheping, Lidong, Daihuang, et al.7 alpha-hydroxy-dehydroepiandrosterone synthesis and activity determination [ J ]. Chinese journal of pharmacy, 2009,43(20): 1596-1598). The medicaments like 7 alpha, 15 alpha-OH-DHEA, 1 alpha-OH-DHEA and the like which are derived and synthesized by DHEA have special pharmacological action and very wide application prospect. Thus, DHEA is an important basic material in steroid drugs, and its industrial synthesis has a significant impact on the development of the entire steroid industry.

In 1956, Rosenkranz et al successfully synthesized DHEA by using dioscin as initial raw material, and formally started the industrialized age of DHEA synthesis. The process of synthesizing DHEA by using tuberous sapogenin is similar and different, and the principle is almost the same and only different from that of selecting different protective agents or oxidizing agents in each step. However, with the development of the times, environmental problems become more and more important problems restricting the development of society and science and technology. The traditional mode of DHEA production requires the consumption of significant diosgenin resources, which are essentially obtained by specialized farming. In addition, the industry generates a large amount of wastewater and other pollutants in the extraction and cracking process of saponin resources, so that the industry faces huge environmental protection pressure. With the advent of an aging society, the consumption of medical resources must be further expanded. This necessarily leads to resource and environmental constraints on DHEA production. Therefore, a new process route is developed to replace the traditional production process.

With the progress of biotechnology, the application of biotechnology to steroid industry is becoming more and more widespread. In the face of resource pressure and environmental pressure, the process of producing 4-AD by degrading phytosterol by using a microbial method has been substantially developed. Leading enterprises in foreign countries and domestic countries have started industrialized scale fermentation production. The process takes a large amount of abandoned and idle sterol resources as raw materials, and can produce the basic 4-AD medicament in the steroid industry on a large scale through large-scale fermentation of engineering bacteria. And 4-AD solves the problem of large-area occupation of cultivated land resources and the problem of environmental pollution. It is expected that the process method is adopted by more enterprises as more engineering bacteria are developed. In view of the high similarity of the structures of 4-AD and DHEA, the method takes 5-AD (II) as a substrate, and 3 beta-hydroxysteroid dehydrogenase is utilized to reduce the 3-carbonyl group to prepare DHEA.

Disclosure of Invention

The invention provides a novel method for preparing DHEA from 5-AD. The invention relates to a method for preparing DHEA by using 3 beta-hydroxy steroid dehydrogenase to reduce 3-carbonyl.

In the synthesis process, the 3 beta-hydroxysteroid dehydrogenase protein sequences are No.6, No.7, No.8, No.9 and No.10 respectively.

The reaction steps of reducing the carbonyl group at the 3-position to beta-hydroxyl under the action of carbonyl reductase to prepare dehydroepiandrosterone (III) are as follows:

a substrate 5-AD, a solvent and a cosolvent form a reaction system, and a biocatalyst and a coenzyme circulating system are added for reaction at the temperature of 25-40 ℃; the time is 4-24 hours, and DHEA is prepared; directly converting by using fermentation liquor, wherein the concentration of a substrate 5-AD is 10 g/L-60 g/L; after the fermentation liquor is concentrated, the substrate concentration can reach 120 g/L. The biocatalyst comprises coenzyme nicotinamide adenine dinucleotide, glucose dehydrogenase or formate dehydrogenase and carbonyl reductase, or crude enzyme powder.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

Example 1

The whole gene synthesis was performed by Shanghai Asahi crown Co.

The gene sequence SEQ No.1 is obtained by optimizing gene WP _084836793.1 from Williamsia sp.1138, the gene sequence SEQ No.2 is obtained by optimizing gene WP _021689360.1 from Novosphingobium tardaugens, the gene No.3 is obtained from Geobacter meterlenduns, and the gene SEQ No.4 is obtained by optimizing gene WP _026035294.1 from Cupriavidus sp.BIS7. The gene is constructed into a corresponding vector. Transforming the prepared recombinant vector into escherichia coli BL21, Rosetta or Origami by a conventional method, constructing genetic engineering bacteria which respectively express 3 beta-hydroxysteroid dehydrogenase SEQ No.5SEQ No.6, SEQ No.7 and SEQ No.8 in a soluble form in the bacteria, and screening out the successfully constructed genetic engineering bacteria, wherein the recombinant bacteria using escherichia coli BL21 as host bacteria have the target proteins with relatively better expression numbers of Ec-1, Ec-2, Ec-3 and Ec-4. Engineering bacteria with the target protein expression amount not less than 20% are used as engineering bacteria strains for production and are preserved in the form of glycerol bacteria or milk freeze-dried strains.

Example 2

50mL of seed liquid is prepared from Ec-1, Ec-2, Ec-3 and Ec-4 respectively, a culture medium is LB liquid culture medium (10 g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl), a single colony of the genetically engineered bacteria is picked by an inoculating loop and inoculated into the culture medium, and the culture is carried out at 37 ℃ and 200rpm overnight. The seed solution cultured overnight was inoculated to a fermentation medium (industrial medium) at an inoculum size of 1%, and cultured at 32 ℃ and 200rpm for 20 hours.

And (3) taking 1mL of fermentation liquor of Ec-1, Ec-2, Ec-3 and Ec-4 respectively, and performing ultrasonic bacteria breaking to detect the activity of the 3 beta-hydroxysteroid dehydrogenase. 3 beta-hydroxysteroid dehydrogenase enzyme activity definition: the enzyme amount required for consuming 1. mu. mol NAD (P) H per minute is 1 enzyme activity unit (U); since the regeneration of coenzyme is required in the reduction process, glucose dehydrogenase or formate dehydrogenase is introduced. The enzyme activity of glucose and formate dehydrogenase is defined as follows: the amount of enzyme required to produce 1. mu. mol NAD (P) H per minute is 1 enzyme activity unit (U).

The enzyme-labeling instrument detects that the 3 beta-hydroxysteroid dehydrogenase activity of the fermentation liquor reaches the data as follows:

numbering	Ec-1	Ec-2	Ec-3	Ec-4
					5AD enzyme activity (U/ml)	3.5	3.5	3.9	3.3

Example 3

Taking 5-AD as a substrate, respectively taking 1mL of fermentation liquor of Ec-1, Ec-2, Ec-3 and Ec-4, 1mL of 2-methyltetrahydrofuran, 30mg of 5-AD, 50mg of glucose and 0.1mg of coenzyme nicotinamide adenine dinucleotide, reacting for 24 hours, extracting by EA in equal volume, and detecting by GC.

Example 5

The 1L system was transformed: 80g of D-glucose, 2g of D-glucose dehydrogenase, NAD⁺0.1g, Ec-1 or Ec-2 or Ec-3 or Ec-4 fermentation broth 500mL, 5-AD 60g dissolved in 500mL 2-methyl tetrahydrofuran, adding the above system and reacting at room temperature. GC detected the progress of the reaction to complete conversion of the substrate. After about 5h, the reaction was extracted three times with EA. The organic phase was dried over anhydrous sodium sulfate and then spin-dried. 62g of crude DHEA was obtained.

Example 6

The 1L system was transformed: 40g of sodium formate, 2g of formate dehydrogenase, NAD⁺0.1g, Ec-1 or Ec-2 or Ec-3 or Ec-4 fermentation broth 500mL, 5-AD 60g dissolved in 500mL 2-methyl tetrahydrofuran, adding the above system and reacting at room temperature. The reaction progress was monitored by GC until complete conversion of the substrate. After about 6h, the reaction was extracted three times with EA. The organic phase was dried over anhydrous sodium sulfate and then spin-dried. 62g of crude DHEA was obtained.

Example 7

The 1L system was transformed: 160g of D-glucose, 3g of D-glucose dehydrogenase, NAD⁺0.1g, Ec-1 or Ec-2 or Ec-3 or Ec-4 fermentation broth 500mL, 5-AD 100g dissolved in 500mL 2-methyl tetrahydrofuran, adding the above system and reacting at room temperature. The reaction progress was monitored by GC until complete conversion of the substrate. After about 15h, the reaction was extracted three times with EA. The organic phase was dried over anhydrous sodium sulfate and then spin-dried. 103g of DHEA crude product is obtained.

Example 8

The 1L system was transformed: 80g of sodium formate, 4g of formate dehydrogenase, NAD⁺0.1g, Ec-1 or Ec-2 or Ec-3 or Ec-4 fermentation broth 500mL, 5-AD 100g dissolved in 500mL 2-methyl tetrahydrofuran, adding the above system and reacting at room temperature. The reaction progress was monitored by GC until complete conversion of the substrate. After about 16h, the reaction was extracted three times with EA. The organic phase was dried over anhydrous sodium sulfate and then spin-dried. 105g of crude DHEA was obtained.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> preparation of Dehydroepiandrosterone (DHEA) using 3 beta-hydroxysteroid dehydrogenase

<130> 2017.10.30

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gttaacgttc tggttaacaa cgctggtatc cagtacttcg ttggtgttga agacatcgaa 300

gctgaacgtg ttatgcgtct gttctctatc aacgttctgg gttctatgct gggtgttaaa 360

accgttgctc cgatcatgaa aaaagctggt gctggtgttg ttatcaacat ctcttctctg 420

gacggtttcc gtggtaccaa cggtatgtct ccgtacgttg cttctaaatg ggctgttcgt 480

ggtctgacca aagctcaggc tctggaactg ggtccggtta tccgtgttgt ttctgttcac 540

ccgggtggtg ttaacacccc gatgggtaac ccgaccggtg acaccggtga agctctgaac 600

gctccgtacg gtcgtgttcc gctgcgtcgt atcggtgaac cgatcgaagt tgctcgtgtt 660

accgctttca tggcttctga cgacgcttct tacgtttctg gttctgaaat cgctgttgac 720

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gccgagggca atgcactggc agccgaactg ggtgatgccg cccgttttta ccaccaggac 180

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gtggatgtgc tggtgaacaa cgccggcatt ctgatgttca agagtctgct ggccaccagt 300

ctggaagagt acgaacgtgt gctgcgcgtg aacctggtgg gtgagtttct gggcattaag 360

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aacgagcgct ttacaaacgt gccgctgcag cgtgttggcg gtccggaaga ggttgcagcc 660

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gacggcggca tgaccatcgg cgtgtattat gaaggctttc cgggtgcccc tggtgttccg 780

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atgggtaaac tggatggcaa agtggccatc gttaccggtg gcgcacgcgg tatgggtcag 60

accaccgcag aagtgtttgt gcaggaaggc gccagcgtgg tgattgtgga cgtgctggat 120

gtggaaggcg aagccctggc aaaacgcctg ggtcgcaata ccatgtacca gcatctggat 180

gtgaccgatg agcagggttg ggaacagctg gtggaaggca ttattgaccg ctacggctgc 240

atcgatatcc tggtgaacaa cgccgccgtg tttttcagca gcccgatcga tgaaacccgc 300

agcgaagcct ttcgtcgcat tctggacatt aacctgatcg gcccgtacct gggtatgaaa 360

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aacggcctgc gtggtagcag cggtagtggt gcctatagcg ccagcaaatg gggcgttcgc 480

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catcctggct ggattgtgac cccgatgaat aacccggacg gcaaaagctt cgaagaagtg 600

aacgccgagc tgaaaattaa gttcccgggc attgccctga gccgtgttgg tcagagcgaa 660

gaaatcgccc gtgccagcct gtttctggca agcgatgaca gcagctacat cagcggcgcc 720

gaactggccg ttgatggtgc ctggagctgt ggcgtgtatc tgcaggataa accgatgcct 780

taa 783

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atgaatcgtc tgcataataa agtggcaatt gtgacaggtg gcgcccgtgg catgggtgca 60

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gccgaaggtg aagcactggc ccgcgaactg ggtggtgcag cacgcttttg caaactggac 180

gtgagtgacc agagcgcctg ggaagcactg gttagcgaaa ccgttgaagc ctttggtcgc 240

attgacgtgc tggtgaacaa tgccgcagtg ctggttttcg gcggtatcac agagctgagc 300

aaacgtgact tcgaacgcgt gatcgccatc aatctggttg gcacctttct gggcattcgt 360

accgtggcac cggtgatgaa acgccagcag gccggcagca tcgtgaacat cagcagcgtt 420

gatggcctgc gtggcgttaa tgcactggcc gcctatgtga gcagcaaatg gggcgtgcgc 480

ggtctgacca aagcagcagc actggaactg ggtctgcacg gcgtgcgcgt gaacagcatt 540

catccgggcg gcgtgaatac cgtgatgagt aatccgaccg gtgccagcgt ggaggaagtg 600

aacaagggct atcagaacgt gccgctgcag cgtgttggtc atccggatga agtggcccgt 660

gccaccctgt ttctggccag cgatgaagca agctactgcc acggtagtga actgagcgtt 720

gacggcggca tggccgcagg cagctattat ccgggtctgc cgggtgcccc gagctaa 777

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Met Gly Arg Leu Glu Gly Lys Thr Ala Ile Val Thr Gly Gly Ala Gln

1 5 10 15

Gly Met Gly Ser Ala Thr Val Arg Val Met Val Glu Glu Gly Ala Lys

20 25 30

Val Val Ile Ala Asp Leu Ala Glu Gln Ala Gly Lys Ser Leu Ala Ala

35 40 45

Glu Leu Gly Asp Ala Ala Ser Phe Cys Arg Leu Asp Val Ser Ser Glu

50 55 60

Ser Asp Trp Gln Lys Val Leu Ala His Thr Leu Glu Val His Gly Thr

65 70 75 80

Val Asn Val Leu Val Asn Asn Ala Gly Ile Gln Tyr Phe Val Gly Val

85 90 95

Glu Asp Ile Glu Ala Glu Arg Val Met Arg Leu Phe Ser Ile Asn Val

100 105 110

Leu Gly Ser Met Leu Gly Val Lys Thr Val Ala Pro Ile Met Lys Lys

115 120 125

Ala Gly Ala Gly Val Val Ile Asn Ile Ser Ser Leu Asp Gly Phe Arg

130 135 140

Gly Thr Asn Gly Met Ser Pro Tyr Val Ala Ser Lys Trp Ala Val Arg

145 150 155 160

Gly Leu Thr Lys Ala Gln Ala Leu Glu Leu Gly Pro Val Ile Arg Val

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Val Ser Val His Pro Gly Gly Val Asn Thr Pro Met Gly Asn Pro Thr

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Arg Arg Ile Gly Glu Pro Ile Glu Val Ala Arg Val Thr Ala Phe Met

210 215 220

Ala Ser Asp Asp Ala Ser Tyr Val Ser Gly Ser Glu Ile Ala Val Asp

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Gly Gly Trp Thr Ala Gly His Tyr His Val Gly Leu Pro Gly Gly Pro

245 250 255

Glu Ala

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<213> Novosphingobium tardaugens

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Met Gly Arg Leu Ser Gly Lys Val Ala Ile Val Thr Gly Gly Ala Arg

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Gly Met Gly Ala Ala Thr Val Arg Leu Phe Val Ala Glu Gly Ala Arg

20 25 30

Val Ala Ile Thr Asp Val Leu Asp Ala Glu Gly Asn Ala Leu Ala Ala

35 40 45

Glu Leu Gly Asp Ala Ala Arg Phe Tyr His Gln Asp Val Thr Ser Glu

50 55 60

Ala Gly Trp Ala Glu Val Val Lys Gln Thr Glu Ala Asp Leu Gly Pro

65 70 75 80

Val Asp Val Leu Val Asn Asn Ala Gly Ile Leu Met Phe Lys Ser Leu

85 90 95

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Val Gly Glu Phe Leu Gly Ile Lys Ala Val Ala Pro Gly Met Ile Ala

115 120 125

Arg Gly Lys Gly Ser Ile Val Asn Val Ser Ser Val Asp Gly Met Lys

130 135 140

Gly Ala Asn Ser Leu Gly Ala Tyr Ala Ser Ser Lys Trp Gly Val Arg

145 150 155 160

Gly Leu Thr Lys Val Ala Ala Met Glu Leu Gly His Arg Gly Ile Arg

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Val Asn Ser Val His Pro Gly Gly Val Asp Thr Ala Met Thr Asn His

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Asn Asn Ala Ser Arg Glu Thr Val Asn Glu Arg Phe Thr Asn Val Pro

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Leu Gln Arg Val Gly Gly Pro Glu Glu Val Ala Ala Ala Ser Leu Phe

210 215 220

Leu Ala Ser Asp Asp Ala Ser Tyr Met Thr Gly Ala Glu Ile Val Val

225 230 235 240

Asp Gly Gly Met Thr Ile Gly Val Tyr Tyr Glu Gly Phe Pro Gly Ala

245 250 255

Pro Gly Val Pro Glu Ala

260

<210> 7

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<212> PRT

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Met Gly Lys Leu Asp Gly Lys Val Ala Ile Val Thr Gly Gly Ala Arg

1 5 10 15

Gly Met Gly Gln Thr Thr Ala Glu Val Phe Val Gln Glu Gly Ala Ser

20 25 30

Val Val Ile Val Asp Val Leu Asp Val Glu Gly Glu Ala Leu Ala Lys

35 40 45

Arg Leu Gly Arg Asn Thr Met Tyr Gln His Leu Asp Val Thr Asp Glu

50 55 60

Gln Gly Trp Glu Gln Leu Val Glu Gly Ile Ile Asp Arg Tyr Gly Cys

65 70 75 80

Ile Asp Ile Leu Val Asn Asn Ala Ala Val Phe Phe Ser Ser Pro Ile

85 90 95

Asp Glu Thr Arg Ser Glu Ala Phe Arg Arg Ile Leu Asp Ile Asn Leu

100 105 110

Ile Gly Pro Tyr Leu Gly Met Lys Ala Val Ile Pro Thr Met Lys Lys

115 120 125

Asn Arg Arg Gly Ser Ile Ile Asn Val Ser Ser Val Asn Gly Leu Arg

130 135 140

Gly Ser Ser Gly Ser Gly Ala Tyr Ser Ala Ser Lys Trp Gly Val Arg

145 150 155 160

Gly Leu Thr Lys Cys Val Ala Met Glu Val Gly Pro Phe Gly Ile Arg

165 170 175

Val Asn Ser Leu His Pro Gly Trp Ile Val Thr Pro Met Asn Asn Pro

180 185 190

Asp Gly Lys Ser Phe Glu Glu Val Asn Ala Glu Leu Lys Ile Lys Phe

195 200 205

Pro Gly Ile Ala Leu Ser Arg Val Gly Gln Ser Glu Glu Ile Ala Arg

210 215 220

Ala Ser Leu Phe Leu Ala Ser Asp Asp Ser Ser Tyr Ile Ser Gly Ala

225 230 235 240

Glu Leu Ala Val Asp Gly Ala Trp Ser Cys Gly Val Tyr Leu Gln Asp

245 250 255

Lys Pro Met Pro

260

<210> 8

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<212> PRT

<213> Cupriavidus sp. BIS7

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Met Asn Arg Leu His Asn Lys Val Ala Ile Val Thr Gly Gly Ala Arg

1 5 10 15

Gly Met Gly Ala Gln Thr Cys Arg Leu Phe Ala Gln Glu Gly Ala His

20 25 30

Val Ile Ile Ala Asp Val Leu Glu Ala Glu Gly Glu Ala Leu Ala Arg

35 40 45

Glu Leu Gly Gly Ala Ala Arg Phe Cys Lys Leu Asp Val Ser Asp Gln

50 55 60

Ser Ala Trp Glu Ala Leu Val Ser Glu Thr Val Glu Ala Phe Gly Arg

65 70 75 80

Ile Asp Val Leu Val Asn Asn Ala Ala Val Leu Val Phe Gly Gly Ile

85 90 95

Thr Glu Leu Ser Lys Arg Asp Phe Glu Arg Val Ile Ala Ile Asn Leu

100 105 110

Val Gly Thr Phe Leu Gly Ile Arg Thr Val Ala Pro Val Met Lys Arg

115 120 125

Gln Gln Ala Gly Ser Ile Val Asn Ile Ser Ser Val Asp Gly Leu Arg

130 135 140

Gly Val Asn Ala Leu Ala Ala Tyr Val Ser Ser Lys Trp Gly Val Arg

145 150 155 160

Gly Leu Thr Lys Ala Ala Ala Leu Glu Leu Gly Leu His Gly Val Arg

165 170 175

Val Asn Ser Ile His Pro Gly Gly Val Asn Thr Val Met Ser Asn Pro

180 185 190

Thr Gly Ala Ser Val Glu Glu Val Asn Lys Gly Tyr Gln Asn Val Pro

195 200 205

Leu Gln Arg Val Gly His Pro Asp Glu Val Ala Arg Ala Thr Leu Phe

210 215 220

Leu Ala Ser Asp Glu Ala Ser Tyr Cys His Gly Ser Glu Leu Ser Val

225 230 235 240

Asp Gly Gly Met Ala Ala Gly Ser Tyr Tyr Pro Gly Leu Pro Gly Ala

245 250 255

Pro Ser

Claims (1)

1. A method of making dehydroepiandrosterone comprising: 3 beta-hydroxy steroid dehydrogenase with amino acid sequence shown as SEQ ID No.6 interacts with substrate 5-AD, and 5-AD is reduced in a proper reduction reaction system to prepare dehydroepiandrosterone DHEA.